PROJECT SUMMARY Increased age is the single most important risk factor for Alzheimer?s Disease (AD). Despite re- cent progress, it remains unclear how aging leads to the impaired proteostasis seen in AD and other neurodegenerative disorders. One possible contributor is age-related impairments in lysosome function. Lysosomal proteases, also known as cathepsins, require an acidic pH in order to function optimally. This acidification may become progressively impaired with age, resulting in impaired protein degrada- tion and potentially enhanced protein aggregation. Despite this, little is known about tissue-specific regulation of lysosomal pH and cathepsin function with age. The long-term goal is to interrogate the basic pathophysiological underpinning of neurodegenerative disease to design rational therapeutics. The overall objective of this application is to utilize systems biology approaches in C. elegans models of aging and neurodegeneration to understand how age and stress affect lysosomal acidification, constituents and activity. The central hypothesis is that age and stress-associated impairments in lysosome function contribute, in a tissue-specific way, to the aberrant protein homeostasis seen in AD and related disorders. The rationale for this work is that through systematic probing and manipulation of lysosomal constituents and pH, one can better understand how the lysosome changes with age, stress and disease. This could lead to new strategies to improve protein homeostasis for treatment or prevention of neurodegenerative diseases. The central hypothesis will be tested through three specific aims: 1) Elucidate the effects of age and stress upon tissue-specific lysosomal pH (pHlys) and protease activity, 2) Determine the basis for age-related lysosome dysfunction via molecular profiling of lysosomes from specific tissues, 3) Identify pathways and molecules that enhance lysosomal acidification. The proposed research is conceptually innovative because of its focus on understanding the tissue-specific changes in lysosomal pH, constituents and function that occur with increasing age and stress. It is also methodologically innovative through its use of a new lysosomal pH and cathepsin D biosensors, development of a new method for lysosome isolation in C. elegans and use of proteomic data to computationally model lysosomal pH. This contribution is significant because age-related lysosome function is an understudied area and these studies could lead to better understanding of how progressive lysosome dysfunction contributes to neurodegenerative disease pathogenesis.